In recent years fluorescence has become a dominant technology in fields such as environmental monitoring, biotechnology (eg drug discovery and cellular imaging) and medicine (eg medical testing and imaging). The greatly enhanced ability to detect specific molecules has also led to rapid advancements in diagnostics. For example, fluorescence detection is widely used in medical testing and DNA analysis because of the high degree of sensitivity obtained using fluorescent techniques. Small numbers of molecules can be detected using fluorescence technology.
Most of the knowledge about fluorescence is based on measurements of the spectroscopic properties of fluorophores that upon excitation, radiate into a homogeneous and non-conducting medium, typically referred to as free space. A fluorophore is like an antenna, which oscillates at high frequency and radiates short wavelengths. Local effects are not usually observed because of the small size of fluorophores relative to the experimental apparatus.
Some of the fluorescence techniques used to detect the presence of molecules include Resonance Energy Transfer (RET), immunofluorescent assays, and fluorescence in situ hybridization. Detection of the molecule of interest is generally limited by the properties of the fluorophore used.
One well-known detection method is Surface Enhanced Raman Scattering (SERS). It is known that the presence of a metallic surface can enhance the Raman signals by factors of 103 to 108 and reports of even larger enhancements have appeared. The presence of a nearby metal film, island or particle can also alter the emission properties of fluorophores.
SERS has been observed for many types of molecules absorbed on the surface of certain metals, such as gold, silver and copper. Similarly, fluorescence signal can also be enhanced many-fold when fluorophores were placed in close proximity to silver particle coated surfaces and silver island films. Enhancement is believed to be due to the interaction of the raman and fluorescence signals with surface plasmon on a metal surface, although the exact mechanisms are not clearly understood. U.S. Ser. No. 10/073,625, which is incorporated by reference in its entirety, discloses compositions and methods for increasing fluorescence intensity of molecules by using metal particles and biomolecules positioned at a distance apart sufficient to adjust intrinsic emission of electromagnetic radiation from the biomolecule in response to an amount of exciting electromagnetic radiation.
Many molecules are not themselves fluorescent. Typically in this case, extrinsic fluorophores are added covalently or non-covalently to allow molecules that do not ordinarily fluoresce or do not fluoresce at useful levels to be detected. However, in some cases, labelling a molecule with an extrinsic fluorophore can alter the activity of the molecule, potentially creating experimental artefacts. For example, labelling a biomolecule may alter the biomolecule such that it loses its biological activity. Problems with current fluorescent techniques stem in part from the low fluorescent intensities of commonly used fluorophores. Additionally, background fluorescence can be significant when using the low wavelength excitation radiation required by some fluorophores or when large quantities of fluorophore are required.
DNA, a biomolecule of great interest to many researchers, ordinarily does not fluoresce at detectable levels. As a result, extrinsic fluorophores are often added to DNA to facilitate the detection of DNA on gels (Benson et al. (1993) Nucleic Acids Res. 21, 5720-5726; Benson et al. (1995) Analytical. Biochem. 231, 247-255), in DNA sequencing (Smith et al. (1986) Nature 321, 674-679; Prober et al. (1987) Science 238, 336-343; Li et al. (1999) Bioconjugate Chem. 10, 241-245), in fluorescence in-situ hybridization (Denijn et al. (1992) APMIS 100, 669-681; Wiegant et al. (2000) Genome Res. 10, 861-865), and for reading of DNA arrays for gene expression (Lipshutz et al. (1999) Nat. Genet. SuppL. 1, 20-24; Ferea et al. (1999) Curr. Opin. Genet. Dev. 9, 715-722).
As DNA sequencing techniques using fluorescent dyes as markers have their maximum emission spectra in the visible range, the DNA is subjected to irradiation in the visible spectra, and visible spectra detectors and light sources are used. Generally photomultiplier tubes are used for detection. As a result, these DNA sequencing techniques have several disadvantages including high costs resulting from the high cost of the lasers used to excite the fluorescent markers which typically emit in the visible region of light spectrum and the high noise to signal ratio due to the background interferences by biomolecules.
Therefore, there remains a need to develop methods of enhancing the fluorescent signals generated by fluorophores to allow the application of fluorescence technology to a variety of analytical, biomedical and material science fields such as physics, chemistry, environmental monitoring and biotechnology.
Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.